Identification of residues within the herpes simplex virus type 1 origin-binding protein that contribute to sequence-specific DNA binding.
Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534-851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780-786 of UL9. Although the corresponding region of the homologous protein encoded by varicell-azoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924.
Stow
N D
ND
MRC Virology Unit, Institute of Virology, Glasgow, United Kingdom. n.stow@vir.gla.ac.uk
Brown
G
G
Cross
A M
AM
Abbotts
A P
AP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000276:immunology / Q000378:metabolism*; D014645:Herpesvirus 3, Human / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1